Methods to enhance and confine expression of genes

ABSTRACT

The present invention provides a novel approach to gene therapy of restricted areas such as tumors. The methods introduced here comprise: (a) placing a gene of interest in a plasmid vector driven by a heat or light inducible promoter; (b) modifying this vector by including a tetracycline responsive fusion protein which acts as a transcriptional activator, thus permitting regulation of gene expression by varying the levels of drug and; (c) modifying this vector by including DNA sequences that reduce or eliminate expression of genes in normal bystander cells. Also provided are a set of vectors for both sustained and regulable expression. There is also presented novel vectors for the gene therapy treatment of local and metastatic breast, ovarian and prostate cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a divisional application of U.S. Ser. No. 09/376,774,filed on Aug. 17, 1999, which claims benefit of priority of provisionalU.S. S. No. 60/096,947, filed Aug. 18, 1998, now abandoned.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the field of genetherapy for cancer. More specifically, the present invention presents amethod of controlling the expression of therapeutically valuable geneproducts via inducible promoters. The present invention provides amethod whereby induced gene expression in the intended cell targets isenhanced and prolonged in a spatially and temporally regulable manner bymeans of heat or light inducible promoters. Moreover, the presentinvention provides a method whereby the background gene expression innon-targeted cells is reduced or eliminated.

[0004] 2. Description of the Related Art

[0005] One of the major obstacles to the success of chemotherapy andradiation therapy for cancer is the difficulty in achievingtumor-specific cell killing. The inability of radiation or cytotoxicchemotherapeutic agents to distinguish between tumor cells and normalcells necessarily limits the dosage that can be applied. As a result,disease relapse due to residual surviving tumor cells is frequentlyobserved.

[0006] The use of gene therapy in cancer treatment presents many of thesame disadvantages as chemotherapy and radiation therapy. Problems withcurrent state-of-the-art gene therapy strategies include the inabilityto deliver the therapeutic gene specifically to the target cells. Thisleads to toxicity in cells that are not the intended targets. Forexample, manipulation of-the p53 gene suppresses the growth of bothtumor cells and normal cells, and intravenous administration of tumornecrosis factor alpha (TNFα) induces systemic toxicity with suchclinical manifestations as fever and hypertension.

[0007] Attempts have been made to overcome these problems. These includesuch strategies as: the use of tissue-specific receptors to direct thegenes to the desired tissues (Kasahara, N., et al., Science,266:1373-1376 (1994)), the use of tissue-specific promoters to limitgene expression to specific tissues (e.g. use of the prostate specificantigen promoter) and the use of heat (Voellmy R., et al., Proc. Natl.Acad. Sci. USA, 82:4949-4953 (1985)) or ionizing radiation inducibleenhancers and promoters (Trainman, R. H., et al., Cell 46: 567-574(1986); Prowess, R., et al., Proc. Natl. Acad. Sci. USA 85, 7206-7210(1988)) to enhance expression of the therapeutic gene in a temporallyand spatially controlled manner. The heat inducible heat shock protein(HSP) promoter has been used to direct the expression of genes such asthe cytokine IL-2.

[0008] Weichselbaum and colleagues were the first to discover theradiation inducible response of the early growth response (Egr-1) genepromoter. Accordingly, they have attempted to direct expression of suchcytotoxic genes as TNF-α to tumor cells to enhance radiation cellkilling by means of this promoter. Previously, systemic administrationof the cytokine TNF-α as an adjuvant to ionizing radiation was initiallyreported to result in enhanced killing in a mouse xenograft tumorsystem. It has since been shown partially effective in human tumors. Theeffect of TNFα appears to be dosage-dependent, as its tumor-killingeffect correlates with its serum concentration. However, systemictoxicity of TNFα restricts the dosage that can be applied and thuslimits the usefulness of the treatment regimen. Attempts have also beenmade to deliver the TNFα gene to tumor cells via adenoviral vectorand/or liposomes. Unfortunately, expression of the TNFα gene is notrestricted to the tumor sites due to the ‘leakiness’ of the promoter.

[0009] In an attempt to localize the level of TNFα to the general areaof radiation exposure and thereby reduce systemic toxicity, Weichselbaumand colleagues employed the radiation inducible Egr-1 promoter toactivate the TNFα gene in situ. Earlier studies showed that theexpression of certain immediate-early genes such as jun/fos and Egr-1are activated in cells exposed to ionizing radiation (Sherman, M. L., etal., Proc. Natl. Acad. Sci. USA, 87: 5663-5666 (1997); Hallahan, D. E.,et al., Proc. Natl. Acad. Sci. USA, 88: 2156-2160 (1991)). By placingthe TNFα gene under the control of the Egr1 promoter (EGRp), theexpression of the TNFα is enhanced in those cells harboring an EGRp-TNFαplasmid when exposed to ionizing radiation. In vivo, the serum level ofTNFα is greatly enhanced (Weichselbaum R. R., et al., Cancer Res. 54:4266-4269 (1994)) within a few hours after irradiation. The combinedtreatment with this plasmid and radiation leads to a partial regressionof a xenografted tumor during the course of the treatment. The level ofTNFα dropped precipitously within 24 hours; further decreases in serumlevel of TNFα coincided with regrowth of the tumors.

[0010] There are several possible reasons for the recurrence of thetumor upon cessation of therapy. The most obvious reason is probably thesame limitation seen with chemotherapy or radiation therapy in general,viz., insufficient dosage levels. A major problem, which limits theamount of TNFα produced, is the weak and transient nature of the Egr-1promoter. This promoter is intrinsically weak, with a maximum of lessthan three-fold increase in expression upon induction. Moreover, theinduced expression is of necessity transient. This, coupled with theweakness of the promoter, permits only a brief exposure of the tumorcells to the TNFα.

[0011] Another factor that limits the production of sufficient dosage ofTNFα, is that not every tumor cell will have taken up the TNFα plasmid.While it has been suggested that repeated administration may help toimprove the treatment outcome, it is not clear if the repeated deliveryof a suboptimal low dosage of TNFα will be useful, the problems posed byan immune response notwithstanding. Although it might be conceivable todeliver larger doses of plasmids, the problem of promoter leakiness hashindered such an approach. It is known that a substantial basal level ofactivity (20-30%) can be detected with the Egr-1 promoter even in theabsence of ionizing radiation (Weichselbaum, et al., supra). This is notsurprising, as the radiation response element, a CArG box, is part ofthe serum response element.

[0012] The HSP promoter is also rather leaky. In the absence of heat,this promoter exhibits a 25-30% background level of expression, notsuitable for most cytotoxic genes. As this level of expression will beharmful to unirradiated normal cells that take up the gene. Hence,administration of this plasmid has been restricted to small doses ofintra-tumoral injections to minimize systemic toxicity.

[0013] Therefore, while it may be advantageous to employ a spatially andtemporally regulated promoter such as the HSP and Egr-1 promoters toenhance specificity of gene expression at the site of heat or radiationtreatment, current versions of those promoters have serious problemsthat restrict their applicability. In order to apply these promoters foruse in cancer therapy, it is necessary to eliminate or greatly reducebackground expression in unheated or unirradiated cells. Ideally, theexpression of cytotoxic genes should be limited to the area of externalstimuli (heat or radiation). Additionally, to ensure a sufficient levelof expression of therapeutic genes, the weak and transient nature ofgene expression driven by these promoters must be improved.

[0014] It is important to note that even when an improved induciblevector system which can restrict the expression of a therapeutic gene tothe area of external stimuli is developed, there is still the problem ofexpression in normal heated or irradiated bystander cells. Thus, it iscritical to be able to further restrict the expression of therapeuticgenes only to the intended targets, e.g., tumor cells.

[0015] The prior art is deficient in the lack of effective means ofinhibiting unwanted toxic side effects of gene therapy treatments forcancer, as well as providing a method for enhancing and sustaining geneexpression in targeted tumor cells in a controllable manner. The presentinvention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

[0016] The current invention provides the composition and methods forthe controlled activation of DNA molecules for gene therapy. Activationof these DNA molecules leads to the production of protein products whichthen may provide opportunities for therapeutic manipulation of cellscontaining said DNA molecules. This may be achieved via alterations incell growth and metabolism of the targeted cells and may include effectson neighboring cells via secretion of therapeutic products. Theinvention offers the options of sustained activation or activationregulable by the application of antibiotics. The invention furtherprovides novel expression vectors for use in gene therapy of local andmetastatic breast, ovarian and prostate cancer.

[0017] An original strategy to confine and enhance therapeutic geneexpression to tumors spatially and temporally is also presented, in theform of an expression vector designed for use in local and metastaticbreast, ovarian and prostate cancer.

[0018] In one embodiment of the present invention, there is provided amethod for sustained and enhanced expression of a gene via activation ofa heat or light inducible promoter. In a modification of this method,heat or light is used to activate the promoter, but continued levels ofgene expression are modulated by concentrations of an antibiotic(tetracycline or its derivatives), acting on a fusion protein with atetracycline-responsive element.

[0019] In yet another embodiment of the present invention, there isprovided a method of constructing the vectors for gene therapyactivation modalities.

[0020] In another embodiment of the present invention, there areprovided improved vectors for reducing background expression in unheatedand unirradiated cells.

[0021] In another embodiment of the present invention, there areprovided improved vectors for reducing expression in heated andirradiated normal bystander cells.

[0022] In another embodiment of the present invention, there areprovided expression vectors for use in gene therapy treatment of localand metastatic breast and ovarian cancer.

[0023] In another embodiment of the present invention, there areprovided expression vectors for use in gene therapy treatment of localand metastatic prostate cancer.

[0024] Other and further aspects, features, and advantages of thepresent invention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] So that the matter in which the above-recited features,advantages and objects of the invention, as well as others which willbecome clear, are attained and can be understood in detail, moreparticular descriptions of the invention briefly summarized above may behad by reference to certain embodiments thereof which are illustrated inthe appended drawings. These drawings form a part of the specification.It is to be noted, however, that the appended drawings illustratepreferred embodiments of the invention and therefore are not to beconsidered limiting in their scope.

[0026]FIG. 1A shows a schematic representation of the plasmid, pDATH-X(Dominant negative, Antisense, TET-ON controllable Heat shock promoterplasmid)-p53, which consists of four cassettes as follows. (1) TET-ON isa fusion of the coding sequences for amino acids 1-207 of thetetracycline (tet) repressor and the C-terminus last 130 amino acidtranscription activation domain of the VP16 protein of the herpessimplex virus (Gossen M., et al., Science, 268:1766-1769 (1995)). InCassette 1, the TET-ON sequence is placed under the control of the HSPand the tet operator binding site and pCMV. (2) HSP is the heat shockpromoter consisting of the heat shock response element (−260 to 30) ofthe human heat shock 70 gene promoter (Voellmy R., et al., Proc. Natl.Acad. Sci. USA 82: 4949-4953 (1985)) linked to the minimal CMV promoter,pCMV (Gossen M., et al., Science, 268:1766-1769 (1995)). In cassette 2,the therapeutic gene, X, is placed under the control of the tetp-pCMVpromoter. (3) tetp is the tet operator consisting of the 19 base pair(bp) inverted repeats of the operator O2 of TN10 (Gossen M, and BujardH., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992)) to which the tetrepressor and TET-ON bind. In cassette 3, antisense TET-ON is placedunder the control of the pCMV promoter. (4) Antisense TET-ON is anantisense sequence consisting of the complementary sequence to the first80 bases of the TET-ON sequence including the ATG. In cassette 4,dominant negative TET-ON is placed under the control of the pCMVpromoter. The Dominant negative TET-ON consists of the tet-repressor butwithout the VP16 transactivation domain, and it is placed under thecontrol of the pCMV promoter. In the absence of heat or light, abackground level of expression of the TET-ON sequence will result due tothe leakiness of the minimal promoter pCMV.

[0027]FIG. 2 depicts the pDATE vector. The plasmid, pDATE-X (Dominantnegative, Antisense, TET-ON controllable EGR promoter expressionplasmid) consists of four cassettes as follows: 1) in cassette 1, theTET-ON sequence is placed under the control of the EGRp, thetetracycline operator binding site and pCMV; 2) in cassette 2, thetherapeutic gene, X, is placed under the control of the tetp-pCMVpromoter; 3) in cassette 3, antisense TET-ON is placed under the controlof the pCMV promoter; and 4) in cassette 4, dominant negative TET-ON isplaced under the control of the pCMV promoter. “TET-ON” is a fusion ofthe coding sequences for amino acids 1-207 of the tet repressor and theC-terminus 130 amino acid transcription activation domain of the VP16protein of the herpes simplex virus. “EGRp” is the radiation induciblepromoter consisting of fragment −425 to +65 of the EGR-1 promotercontaining four copies of the CArG domain. “ptet” is the tet operatorconsisting of the 19 bp inverted repeats of the operator O2 of TN10 towhich the tet repressor and TET-ON bind, linked to the minimal CMVpromoter, pCMV. “Antisense Tet-On” is a sequence consisting of thecomplementary sequence to the first 80 bases of the TET-ON sequenceincluding the ATG. “Dominant negative TET-ON” consists of the codingsequences for amino acids 1-207 of the tet repressor placed under thecontrol of the pCMV promoter. “M” is the chicken lysosomal matrixattachment site to isolate the position effects of each of thecassettes.

[0028]FIG. 3 depicts the structure of the pRIBs-X (Radiation-Inducible,Breast-specific Promoter) expression vector. The pRIBS vector iscomprised of four cassettes. Gene cassette 1 differs from previouslydescribed vectors only in that it contains “Gal-DBD-mx” which is afusion open reading frame (ORF) encoding the N-terminus (amino acids1-147) DNA-binding domain of the yeast GAL4 protein (Gal-DBD) fused tothe basis helix-loop-helix-leucine zipper (bHLHLZ) domain of Max (mx,amino acids 8-112) followed by SV40 poly A. Gene cassette 2 is comprisedof the minimal CMV promoter (mCMVp), “antisense Gal-DBD-mx”, which is anantisense construct complementary to the Gal-DBD-mx sequence, “IRES”,which is an internal ribosomal entry site and “Gal-DBD” which competeswith the Gal-DBD-mx for the pGAL binding site. Gene cassette 3 iscomprised of “VP16-TA-mc” which is a fusion open reading frame encodingat the N-terminus the first 11 amino acids of Gal4 (amino acids 1-147),followed by the nuclear localization signal of the SV40 large T antigen,the 130 amino acid C-terminus transactivation domain of the herpessimplex viral protein VP16, the bHLHLZ domain of c-Myc (amino acids350-439), followed by SV40 polyA. The resulting fusion gene, VP16TA-mc,is placed under the control of the c-erbB-2 promoter “perbB2” up to thefirst ATG. Gene cassette 4 contains “GALp”, consisting of five copies ofa 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed underthe control of the GALp-ptet promoter and the therapeutic gene, X, islinked to the TET-ON via an IRES; Gene cassette 5 contains an antisenseTET-ON which is a sequence consisting of the complementary sequence tothe first 80 bases of the TET-ON sequence including the ATG, placedunder the control of the pCMV promoter. Gene cassette 6 contains adominant negative TET-ON consisting of the coding sequences for aminoacids 1-207 of the tet repressor placed under the control of the pCMVpromoter.

[0029]FIG. 4 shows the structure of the pRIPS-GFP (Radiation-Inducible,Prostate-specific Promoter) expression vector. The pRIPS vector iscomprised of six cassettes. Gene cassette 1 differs from previouslydescribed vectors only in that it contains “Gal-DBD-mx” which is afusion open reading frame encoding the N-terminus (amino acids 1-147)DNA-binding domain of the yeast GAL4 protein (Gal-DBD) fused to thebasis helix-loop-helix-leucine zipper (bHLHLZ) domain of Max (mx, aminoacids 8-112) followed by SV40 poly A. Gene cassette 2 is comprised ofthe minimal CMV promoter (mCMVp), “antisense Gal-DBD-mx”, which is anantisense construct complementary to the Gal-DBD-mx sequence, “IRES”,which is an internal ribosomal entry site and “Gal-DBD” which competeswith the Gal-DBD-mx for the pGAL binding site. Gene cassette 3 iscomprised of “VP16-TA-mc” which is a fusion open reading frame encodingat the N-terminus the first 11 amino acids of Gal4 (amino acids 1-147),followed by the nuclear localization signal of the SV40 large T antigen,the 130 amino acid C-terminus transactivation domain of the herpessimplex viral protein VP16, the bHLHLZ domain of c-Myc (amino acids350-439), followed by SV40 polyA. The resulting fusion gene, VP16-TA-mc,is placed under the control of the probasin gene promoter “pProbasin” upto the first ATG. Gene cassette 4 contains “GALp”, consisting of fivecopies of a 17-mer DNA-binding site for Gal4. The TET-ON sequence isplaced under the control of the GALp-ptet promoter and the therapeuticgene, X, is linked to the TET-ON via an IRES; Gene cassette 5 contains an antisense TET-ON which is a sequence consisting of the complementarysequence to the first 80 bases of the TET-ON sequence including the ATG,placed under the control of the pCMV promoter. Gene cassette 6 containsa dominant negative TET-ON consisting of the coding sequences for aminoacids 1-207 of the tet repressor placed under the control of the pCMVpromoter.

[0030]FIG. 5 is a schematic representation of the mode of action ofpRIBS-GFP.

[0031]FIG. 6 illustrates the leakiness of the HSP promoter. Itsummarizes the results of testing the heat inducible system containingthe hsp70 promoter in the expression of therapeutic genes, p53 and TNFα.

[0032]FIG. 6A shows the plasmid construct for the two genes, p53 andTNFα.

[0033]FIG. 6B depicts p53 transcriptional activity. To analyze theinducibility of the hsp promoter, the plasmid pHSP.3p53CD1 or thecontrol pHSP.3 vector alone was cotransfected with Post-2-CAT(containing a CAT coding sequence linked to a consensus p53 bindingsite) into the human ovarian carcinoma cell line SKOV3 which has ahomozygous deletion of p53. At 36 hours after transfection, cells wereeither heated or unheated. CAT activity was measured 24 hours later.Little or no activity is seen with the SKOV3 parental untransfectedcells (lane 2, heated; lane 1, unheated). Similarly, with the pHSP.3vector alone, there is no activity with or without heat (lanes 3 and 4).With the pHSP.3p53 plasmid, there is a high level of CAT activity seenat 24 hrs after heating (lane 6). However, even without heating (lane5), there is a substantial level of p53 expression (about 25%).

[0034]FIG. 7 depicts the induction of TNFα by heat or photodynamictherapy (PDT). The coding sequence of TNFα was subcloned into theplasmid pHSP.3 and transfected into SKOV3 cells. Stable colonies wereisolated by selection in G418. Cells were either heated at 45° C. oruntreated. At 6 hours after treatment, the level of TNFα in the mediumwas measured with a Genzyme TNFα ELISA kit. TNFα shown to be inducedfour-fold by heat and three-fold by PDT and secreted. However,background expression was substantial (27%).

[0035]FIG. 8 shows the expression kinetics of p53 in the H358 lungcarcinoma cell line by the feed-forward reaction, where a,b,c,d and erepresent the levels of p53 reached at 10 hours after the feed-forwardreaction. Six hours after heat shock, transfected cells were treatedwith different doses of doxycycline. At various time points after theaddition of doxycycline, the cells were stained with a p53 antibody. Foreach point, the digital images of fifty immunostained cells werecaptured using a Nikon microscope. The amount of protein expressed ineach cell is proportional to the intensity of staining, expressed asI=1/T (where T is a measure of the transmitted light/unit area. Thisplot shows the results of one such experiment using 0.01-0.1 μg/mldoxycycline.

[0036]FIG. 9 depicts the pHIBS-X (Heat-Inducible, Breast-specificPromoter) expression vector. The pHIBS vector is comprised of sixcassettes. Gene cassette 1 differs from the vectors described above onlyin that it contains “Gal-DBD-mx” which is a fusion open reading frameencoding the N-terminus (amino acids 1-147) DNA-binding domain of theyeast GAL4 protein (Gal-DBD) fused to the basis helix-loop-helix-leucinezipper (bHLHLZ) domain of Max (mx, amino acids 8-112) followed by SV40poly A. The resulting fusion gene GAL-DBD-mx is controlled by the heatinducible HSP promoter. Gene cassette 2 is comprised of the minimal CMVpromoter (mCMVp), “antisense Gal-DBD-mx”, which is an antisenseconstruct complementary to the Gal-DBD-mx sequence, “IRES”, which is aninternal ribosomal entry site and “Gal-DBD” which competes with theGal-DBD-mx for the pGAL binding site. Gene cassette 3 is comprised of“VP16-TA-mc” which is a fusion open reading frame encoding at theN-terminus the first 11 amino acids of Gal4 (amino acids 1-147),followed by the nuclear localization signal of the SV40 large T antigen,the 130 amino acid C-terminus transactivation domain of the herpessimplex viral protein VP16, the bHLHLZ domain of c-Myc (amino acids350-439), followed by SV40 polyA. The resulting fusion gene, VP16TA-mc,is placed under the control of the c-erbB-2 promoter “perbB2” up to thefirst ATG. Gene cassette 4 contains “GALp”, consisting of five copies ofa 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed underthe control of the GALp-ptet promoter and the therapeutic gene, X, islinked to the TET-ON via an IRES; Gene cassette 5 contains an antisenseTET-ON which is a sequence consisting of the complementary sequence tothe first 80 bases of the TET-ON sequence including the ATG, placedunder the control of the pCMV promoter. Gene cassette 6 contains adominant negative TET-ON consisting of the coding sequences for aminoacids 1-207 of the tet repressor placed under the control of the pCMVpromoter.

[0037]FIG. 10 illustrates the structure of the pHIPs-GFP(Heat-Inducible, Prostate-specific Promoter) expression vector. ThepHIPS vector is comprised of six cassettes. Gene cassette 1 differs frompreviously described vectors only in that it contains “Gal-DBD-mx” whichis a fusion open reading frame encoding the N-terminus (amino acids1-147) DNA-binding domain of the yeast GAL4 protein (Gal-DBD) fused tothe basis helix-loop-helix-leucine zipper (bHLHLZ) domain of Max (mx,amino acids 8-112) followed by SV40 poly A. The resulting fusion geneGAL-DBD-mx is controlled by the heat inducible HSP promoter. Genecassette 2 is comprised of the minimal CMV promoter (mCMVp), “antisenseGal-DBD-mx”, which is an antisense construct complementary to theGal-DBD-mx sequence, “IRES”, which is an internal ribosomal entry siteand “Gal-DBD” which competes with the Gal-DBD-mx for the pGAL bindingsite. Gene cassette 3 is comprised of “VP16-TA-mc” which is a fusionopen reading frame encoding at the N-terminus the first 11 amino acidsof Gal4 (amino acids 1-147), followed by the nuclear localization signalof the SV40 large T antigen, the 130 amino acid C-terminustransactivation domain of the herpes simplex viral protein VP16, thebHLHLZ domain of c-Myc (amino acids 350-439), followed by SV40 polyA.The resulting fusion gene, VP16TA-mc, is placed under the control of theprobasin gene promoter (pProbasin) up to the first ATG. Gene cassette 4contains “GALp”, consisting of five copies of a 17-mer DNA-binding sitefor Gal4. The TET-ON sequence is placed under the control of theGALp-ptet promoter and the therapeutic gene, X, is linked to the TET-ONvia an IRES; Gene cassette 5 contains an antisense TET-ON which is asequence consisting of the complementary sequence to the first 80 basesof the TET-ON sequence including the ATG, placed under the control ofthe pCMV promoter. Gene cassette 6 contains a dominant negative TET-ONconsisting of the coding sequences for amino acids 1-207 of the tetrepressor placed under the control of the pCMV promoter.

DETAILED DESCRIPTION OF THE INVENTION

[0038] As used herein, the term “heat” is to mean heat energy generatedby any means, including microwaves.

[0039] As used herein, the term “light” is to mean light energy withfrequencies in the visible as well as the invisible spectrum, includingionizing radiation generated by any means. This would include aradiation source such as radionuclides capable of emitting gamma and orbeta particles, or by a linear accelerator.

[0040] In accordance with the present invention, there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: APractical Approach,” Volumes I and II (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcriptionand Translation” [B. D. Hames & S. J. Higgins eds. (1984)]; “Animal CellCulture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes”[IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning”(1984).

[0041] The present invention is directed towards a new method of genetherapy for confined areas such as tumors. In accordance with theabove-mentioned object there is provided a mechanism for bothconstitutively active and regulable gene expression via plasmidscontaining elements which are heat and or light activated and responsiveto presence and concentration of antibiotic (tetracycline and itsderivatives). In regulating gene expression, heat or light initiates theexpression, but the gene is constitutively expressed only in thepresence of the antibiotic (tetracycline and its derivatives).Concentration of the antibiotic controls the level and duration of thegene expression.

[0042] For the confinement of gene expression to tumor cells, there areprovided two mechanisms for the suppression of gene expression in normalcells that are bystander targets of heat or radiation. In the instanceof normal cells not exposed to heat or light, which inadvertently takeup the plasmid, expression of the therapeutic gene due to backgroundactivity of the promoter is suppressed by the constitutive expression ofantisense and dominant negative DNA sequences to the heat or lightinducible, antibiotic dependent transcriptional activator built into theplasmid. In the instance whereby normal cells that take up the plasmidare then exposed to heat or light, there is an additional mechanism forpreventing the expression of the therapeutic gene. This is achieved bythe use of a modified ‘two hybrid’ system where the antibiotic dependenttranscriptional activator is itself under the control of both theexpression of tissue-specific transcriptional activators and theexposure to heat or light. Expression of the therapeutic gene istherefore found only in cells that have been both exposed to heat orlight and that express tissue-specific transcription factors.

[0043] In one embodiment of the present invention, there is provided arecombinant vector, pDATH-X (dominant negative, Antisense, TET-ONcontrollable Heat shock promoter plasmid), for the purpose of reducingbackground levels of expression. This vector is comprised of thecassettes: (a) a fusion of the coding sequences for amino acids 1-207 ofthe tetracycline repressor and the C-terminus last 130 amino acidtranscription activation domain of the VP16 protein of the herpessimplex virus; (b) a heat shock promoter consisting of heat shockresponse elements (−260 to 30) of the human heat shock 70 gene promoterlinked to the minimal cytomegalovirus promoter, pCMV; (c) a tet operatorconsisting of the 19 bp inverted repeats of the operator O2 of TN10 towhich the tet repressor and TET-On bind; and (d) an antisense sequenceconsisting of the complementary sequence to the first 80 bases of theTET-On sequence including the ATG.

[0044] In another embodiment of the present invention provides a methodof achieving sustained expression of a gene under control of a heat orlight inducible promoter, comprising the step of: introducing the vectorcontaining said gene into the host organism; and applying heat or lightenergy. In another embodiment of the invention, said host organism is ahuman.

[0045] In yet another embodiment of the invention, there is provided arecombinant vector, pDATE-X (Dominant negative, Antisense, TET-ONcontrollable EGR promoter expression plasmid), said vector comprisingthe cassettes: (a) in cassette 1, the TET-ON sequence is placed underthe control of the EGRp, the tetracycline operator binding site andpCMV; (b) in cassette 2, the therapeutic gene X, is placed under thecontrol of the tetp-pCMV promoter; (c) in cassette 3, antisense TET-ONis placed under the control of the pCMV promoter; and (d) in cassette 4,dominant negative TET-ON is placed under the control of the pCMVpromoter.

[0046] Another embodiment of the present invention provides arecombinant vector, pRIBs-X, (Radiation-Inducible, Breast-specificPromoter) expression vector, said vector comprising the cassettes: (a)cassette 1 contains “Gal-DBD-mx” which is a fusion open reading frameencoding the N-terminus (amino acids 1-147) DNA-binding domain of theyeast GAL4 protein (Gal-DBD) fused to the basic helix-loop-helix-leucinezipper domain of Max (amino acids 8-112) followed by SV40 poly A—theresulting fusion gene GAL-DBD-mx is controlled by the radiationinducible Egr-1 promoter; (b) cassette 2 is comprised of the minimal CMVpromoter, “antisense Gal-DBD-mx”, which is an antisense constructcomplementary to the Gal-DBD-mx sequence, “IRES”, which is an internalribosomal entry site and “Gal-DBD” which competes with the Gal-DBD-mxfor the pGAL binding site; (c) cassette 3 is comprised of “VP16-TA-mc”which is a fusion open reading frame encoding at the N-terminus thefirst 11 amino acids of Gal4 (amino acids 1-147), followed by thenuclear localization signal of the SV40 large T antigen, the 130 aminoacid C-terminus transactivation domain of the herpes simplex viralprotein VP16, the basic helix-loop-helix-leucine zipper domain of c-Myc(amino acids 350-439), followed by SV40 polyA—the resulting fusion gene,VP16-TA-mc, placed under the control of the c-erbB2 promoter “perB2” upto the first ATG; (d) cassette 4 contains “Galp”, five copies of a17-merDNA-binding site for Gal4. The TET-ON sequence is placed under thecontrol of the GAPp-ptet promoter and the therapeutic gene, X, is linkedto the TET-IN via an IRES; (e) cassette contains an antisense TET-ONwhich is a sequence consisting of the complementary sequence to thefirst 80 bases of the TET-ON sequence including the ATG, placed underthe control of the pCMV promoter; and (f) cassette 6 contains a dominantnegative TET-ON consisting of the coding sequences for amino acids1-207.

[0047] There are further provided variants of the preceding vectors,wherein the perbB2 promoter is replaced with the whey acidic proteinpromoter or the stromelysin 3 promoter.

[0048] Another embodiment of the invention provides a method for thetreatment of local and metastatic breast and ovarian cancer comprising:administration to the patient a pRIBs-X expression vector (or a variantthereof) containing a cytotoxic gene. A representative cytotoxic gene istumor necrosis factor alpha.

[0049] The present invention is also directed to a recombinant pRIPs-X(Radiation-Inducible, Prostate-specific Promoter) expression vector,said vector comprising the cassettes: (a) cassette 1 contains“Gal-DBD-mx” which is a fusion open reading frame encoding theN-terminus (amino acids 1-147) DNA-binding domain of the yeast GAL4protein fused to the basic helix-loop-helix leucine zipper domain of Max(amino acids 8-112) followed by SV40 polyA—the resulting fusion geneGAL-DBD-mx is controlled by the radiation inducible Egr-1 promoter; (b)cassette 2 is comprised of the minimal CMV promoter, antisenseGal-DBD-mx, which is an antisense construct complementary to theGal-DBD-mx sequence, IRES, which is an internal ribosomal entry site andGal-DBD which competes with the Gal-DBD-mx for the pGAL binding site;(c) cassette 3 is comprised of “VP16-TA-mc”, a fusion open reading frameencoding at the N-terminus the first 11 amino acids of Gal4, followed bythe nuclear localization signal of the SV40 large T antigen, the 130amino acid C-terminus transactivation domain of the herpes simplex viralprotein VP16, the basic helix-loop-helix leucine zipper domain of c-Myc(amino acids 350-439), followed by SV40 polyA—the resulting fusion gene,VP16-TA-mc, is placed under the control of the probasin gene promoter“pProbasin” up to the first ATG; (d) cassette 4 contains GALp, fivecopies of the 17-mer DNA-binding site for Gal4. The TET-ON sequence isplaced under the control of the GALp-ptet promoter and the therapeuticgene, X, is linked to the TET-ON via an internal ribosomal entry site;(e) cassette 5 contains an antisense TET-ON which is a sequenceconsisting of the complementary sequence to the first 80 bases of theTET-ON sequence including the ATG, placed under the control of the pCMVpromoter; and (f) cassette 6 contains a dominant negative TET-ONconsisting of the coding sequence for amino acids 1-207. A variant ofthe preceding vector is also contemplated, wherein the probasin promoteris replaced with the prostate specific antigen promoter.

[0050] Another embodiment of the invention provides a method for thetreatment of local and metastatic prostate cancer comprising:administration to the patient a pRIPs-X expression vector (or a variantthereof) containing a cytotoxic gene. A representative cytotoxic gene istumor necrosis factor alpha.

[0051] In yet another embodiment of the present invention, there isprovided a recombinant expression vector, pHIBs-X (Heat Inducible,Breast-specific promoter), said vector comprising the cassettes: (a)cassette 1 contains Gal-DBD-mx which is a fusion open reading frameencoding the N-terminus (amino acids 1-147) DNA-binding domain of theyeast GAL4 protein fused to the basic helix-loop-helix leucine zipperdomain of Max (amino acids 8-112) followed by SV40 polyA—the resultingfusion gene GAL-DBD-mx is controlled by the heat inducible heat shockprotein promoter; (b) cassette 2 is comprised of the minimal CMVpromoter, antisense Gal-DBD-mx, a construct complementary to theGal-DBD-mx sequence, an internal ribosomal entry site and Gal-DBD, whichcompetes with the Gal-DBD-mx for the pGAL binding site; (c) cassette 3is comprised of “VP16-TA-mc” which is a fusion open reading frameencoding at the N-terminus the first 11 amino acids (amino acids 1-147),followed by the nuclear localization signal of the SV40 large T antigen,the 130 amino acid C-terminus transactivation domain of the herpessimplex viral protein VP16, the basic helix-loop-helix leucine zipperdomain of c-Myc (amino acids 350-439), followed by SV40 polyA—theresulting fusion gene VP16-TA-mc is placed under the control of thec-erbB2 gene promoter “perbB2” up to the first ATG; (d) cassette 4contains GALp, five copies of a 17-mer DNA-binding site for Gal4. TheTET-ON sequence is placed under the control of the GALp-ptet promoterand the therapeutic gene, X, is linked to the TET-ON via an internalribosomal entry site; (e) cassette 5 contains an antisense TET-ON whichis a sequence consisting of the complementary sequence to the first 80bases of the TET-ON sequence including the ATG, placed under the controlof the pCMV promoter; and (f) cassette 6 contains a dominant negativeTET-ON consisting of the coding sequences for amino acids 1-207.Variants of the preceding vector are contemplated, wherein the perbB2promoter is replaced with the whey acidic protein promoter or thestromelysin 3 promoter.

[0052] The present invention is further directed to a method for thetreatment of local and metastatic breast and ovarian cancer comprising:administration to the patient a pHIBs-X expression vector (or a variantthereof) containing a therapeutic gene. A representative therapeuticgene is tumor necrosis factor alpha.

[0053] Another embodiment of the invention provides a recombinantvector, pHIPs-X (Heat-Inducible, Prostate-specific Promoter), saidvector comprising the cassettes: (a) cassette 1 contains Gal-DBD-mxwhich is a fusion open reading frame encoding the N-terminus (aminoacids 1-147) DNA-binding domain of the yeast GAL4 protein fused to thebasic helix-loop-helix leucine zipper domain of Max (amino acids 8-112)followed by SV40 polyA—the resulting fusion gene GAL-DBD-mx iscontrolled by the heat inducible heat shock protein promoter; (b)cassette 2 is comprised of the minimal CMV promoter (mCMVp), antisenseGal-DBD-mx, a construct complementary to the Gal-DBD-mx sequence, aninternal ribosomal entry site and Gal-DBD, which competes with theGal-DBD-mx for the pGAL binding site; (c) cassette 3 is comprised of“VP16-TA-mc”, a fusion open reading frame encoding at the N-terminus thefirst 11 amino acids of Gal4, followed by the nuclear localizationsignal of the SV40 large T antigen, the 130 amino acid C-terminustransactivation domain of the herpes simplex viral protein VP16, thebasic helix-loop-helix leucine zipper domain of c-Myc (amino acids350-439), followed by SV40 polyA—the resulting fusion gene, VP16-TA-mc,is placed under the control of the probasin gene promoter “pProbasin” upto the first ATG; (d) cassette 4 contains GALp, five copies of a 17-merDNA-binding site for Gal4. The TET-ON sequence is placed under thecontrol of the GALp-ptet promoter and the therapeutic gene, X, is linkedto the TET-ON via an internal ribosomal entry site; (e) cassette 5contains an antisense TET-ON which is a sequence consisting of thecomplementary sequence to the first 80 bases of the TET-ON sequenceincluding the ATG, placed under the control of the pCMV promoter; and(f) cassette 6 contains a dominant negative TET-ON consisting of thecoding sequences for amino acids 1-207. A variant of the precedingvector is contemplated, wherein the probasin promoter is replaced withthe prostate-specific antigen promoter.

[0054] In another embodiment of the invention, there is provided amethod for the treatment of local and metastatic prostate cancercomprising: administration to the patient a pHIPs-X vector (or a variantthereof) containing a therapeutic gene. representative therapeutic geneis tumor necrosis alpha.

[0055] It is specifically contemplated that pharmaceutical compositionsof the present invention may be prepared for the purpose of genetherapy. In such a case, the composition comprises a vector of thepresent invention and a pharmaceutically acceptable carrier. A personhaving ordinary skill in the art of cancer chemotherapy would readily beable to determine, without undue experimentation, appropriate dosagesand routes of administration. For gene therapy, the gene of interestcontained in one of the plasmid vectors of the present invention, couldbe delivered to the target cell via a viral vector or liposome.

[0056] The level of ordinary skill of the average scientist in the areaof molecular cancer biology has increased substantially in recent years.A person having ordinary skill in this art would readily be able toconstruct and utilize the plasmids for this novel approach to genetherapy given the teachings of the present specification.

[0057] The following examples are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion.

EXAMPLE 1

[0058] The pDATE Vector: Structure and Mode of Action

[0059]FIG. 2 is a schematic depiction of the pDATE vector. The pDATE-Xplasmid functions via a feed-forward reaction to amplify the expressionof TET-ON and X. In the absence of radiation, background expression dueto leakiness of the EGRp will result in the synthesis of TET-ON mRNA.Translation of this mRNA is reduced by the concomitant expression ofantisense TET-ON RNA. Moreover, leaked-through translated TET-ON proteinis inactive without tetracycline. In the presence of tetracycline, theleaked (translated) TET-ON protein becomes active, but the feed-forwardreaction is prevented by the constitutively expressed dominant negativeTET-ON protein which competes for the same DNA binding site of the ptetpromoter.

[0060] Two chicken lysosomal matrix attachment sites (MAR) are insertedto isolate the position effects of the cassettes (McKnight, R. A., etal., Mol. Reprod. & Dev., 44:179-184 (1996)). While they may beunnecessary when the antisense and dominant negative TET-ON expressionsare driven by the minimal CMV promoter, MARs may be needed if strongerpromoters like the human 3 actin promoter are to drive their expression.

[0061] When cells harboring the pDATE-X are exposed to radiation, aninitial burst of TET-ON transcription occurs, leading to the synthesisof 2-4-fold above background level of TET-ON in greater excess than thedominant negative TET-ON. This excess TET-ON protein, in the presence oftetracycline, then binds to the tetp promoters to which the codingsequence of both TET-ON and X are linked and engages in a feed-forwardreaction. This reaction is controlled by the level of tetracycline. Assuch, X expression is elevated and the duration lengthened untiltetracycline is removed, at which point the half-life of the TET-ONprotein will determine how long the feed-forward reaction can berestarted using tetracycline without further radiation exposure.

[0062] This vector makes use of a feed-forward reaction to achieve andmaintain a high level of inducible gene expression. This feed-forwardfeature overcomes the transient nature and weakness of the induciblepromoter. When the feed-forward reaction is limited to a few hours,there is a large difference in the level of TET-ON achieved in heatedand unheated cells. It is thus possible to adjust the difference in thelevel of amplified TET-ON in irradiated and unirradiated cells byenhancing the former with the alternate addition and removal oftetracycline. However, while the addition and removal of tetracyclinecan be precisely controlled in cell culture, it is difficult to do so invivo due to the heterogeneity of tetracycline level in tissues and thevariation in the absorption and removal of tetracycline in vivo indifferent individuals. Thus, it is critical to minimize the leak throughexpression of TET-ON with antisense and dominant negative cassettes sothat the feed-forward reaction does not significantly amplify its levelin unirradiated cells.

[0063] If necessary, the action of this vector can be further fine-tunedby replacing the pCMV minimal promoter with a much stronger promotersuch as the human β actin promoter to drive the expression of theantisense and the dominant negative TET-ON. In addition, the copynumbers of the antisense and the dominant negative coding sequences canbe increased.

[0064] For in vivo induction of TET-ON expression, oxytetracycline willbe used because of its short in vivo half-life. In humans, after asingle oral dose peak plasma concentration of oxytetracycline is reachedat 2-4 hours (see, e.g., Goodman & Gilman's The Pharmacological Basis ofTherapeutics). The level of TET-ON expression as a function ofoxytetracycline concentration can thus be monitored. Oxytetracycline isshort acting with an in vivo half-life of only 9 hours (versusdoxycycline which has a half-life of 18 hours). At the end of 24 hours,the oxytetracycline level is reduced to <25% of input (about 10-30 % arenever absorbed and are excreted in the active form).

EXAMPLE 2

[0065] The pDATH Vector: Structure and Mode of Action

[0066]FIG. 1 is a schematic depiction of the pDATH-X vector. This vectoroperates in identical fashion to the pDATE-X vector, except that theEgr-1 promoter is replaced with the HSP promoter and that heat is usedin place of light/ionizing radiation.

EXAMPLE 3

[0067] Verification of the Concept of Amplifiable and SustainedExpression of TET-On and p53 With the Feed-forward Inducible Promoter

[0068] To further validate the concept of heat inducible, tetracyclinefeed-forward amplification of gene expression, two plasmids wereconstructed. The plasmid “ptet-splice p53wt” was constructed bysubcloning a wild-type p53 cDNA into the ptet-splice vector (Gibco BRL)which places p53 under the control of the tetp promoter (consists of theregulatory sequences from the tetracycline-resistance operon upstream ofa minimal hCMV promoter). The plasmid “HSP-tetp-TET-ON” was constructedby replacing the CMV promoter in ptet-on (Clontech) with 300 bp of thehuman heat shock protein promoter and the tetp promoter.

[0069] H358, a non-small cell lung carcinoma cell line with a homozygousdeletion of p53, was grown in RPMI+10% fetal calf serum. 10⁷exponentially growing cells were cotransfected with 50 μg of“ptet-splice p53wt” and 10 μg of “HSP-tetp-TET-ON” by electroporationusing a BRL cell-Porator at 1180 μF and 240 V in 0.8 ml RPMI+6 mMglucose. Transfected cells were plated out at 25% confluence for 36hours and then half of them were heat-shocked at 45° C. for twentyminutes. Six hours after heat shock, cells were treated with differentdoses of doxycycline. At various time points after the addition ofdoxycycline, cells were stained immunohistochemically with themonoclonal p53 antibody DO-1 (Santa Cruz Biologicals) using animmunoperoxidase cell staining kit (Vector) and diaminobenzidine (DAB).For each point, the digital images of fifty immunostained cells werecaptured using a Nikon microscope. The amount of protein expressed ineach cell is proportional to the intensity of staining which wasexpressed as I=1/T, where T is a measure of the transmitted light/unitarea. Results of one such experiment at 0.01-0.1 μg/ml of doxycyclineare shown in FIG. 8.

[0070] When 0.1 μg/ml of doxycycline was added at 6 hours after heating(when the level of induced TET-ON should have been at its peak), morethan 12 fold amplification of p53 was reached in 10 hours (curves a andb, FIG. 8). During this time, doxycycline also started a feed-forwardreaction in the unheated cells as indicated by the substantial level ofTET-ON. However, since the amplification started off from a lower level,the amplified level of TET-ON at 10 hours reached only a low level(curves c and d, FIG. 8).

[0071] It is possible to regulate the level of induced p53 in thefeed-forward system with an alternate regimen of tetracycline additionand removal. In the time it takes for TET-ON (e.g. FIG. 8 level [c]) inthe unheated cells to decline back to background level [e] afterremoval, the level of TET-ON in the heated cells, [a], would havedeclined by a similar proportion (which is equal to [c]-[e]). However,since this level ([a]-[c]-[e]) is much higher than in the unheated cells[e], the addition of tetracycline will re-start the feed-forwardreaction for the heated cells from a much higher level ([a]-([c]-[e])).As such, the level of background p53 in unheated cells can be kept at orbelow the low level reached at 10 hours ([c]) whereas the p53 level inheated cells will continue to escalate. Thus, while the TNFα and p53expression driven by the HSP directly is transient, the expressiondriven by the feed-forward system is on for as long as tetracycline isavailable. Since the regimen of tetracycline addition in vivo will bedetermined by the decay rate of tetracycline in vivo, it is important toknow the half-life of the TET-ON in tumor cells.

[0072] In vivo, the pharmacokinetics of tetracycline is heterogeneousfor different tissues. Preferential concentration of tetracycline inspecific tissues will lead to higher background expression of TET-ON insome tissues. For example, in humans, 10-35% of oxytetracycline isremoved via the kidney, a substantial amount of which is excreted in theactive form. Therefore, it is desirable to minimize the backgroundexpression levels at the onset to prevent run away amplification in theunintended tissues. The pDATE and pDATH inducible systems use aconstitutively expressed antisense TET-ON to suppress the backgroundlevel of TET-ON translation and a dominant negative TET-ON to competewith leak-through expressed TET-ON to suppress the backgroundexpression. With the suppressed background, the timing of tetracyclineaddition is only affected by the desired level and duration of theexpression of the therapeutic genes and not by the need to suppress thelevel of background expression in normal unirradiated cells.

EXAMPLE 4

[0073] Reduction in Background Levels of Expression

[0074] Employing the 300 bp HSP promoter, the background level ofexpression without heat or light is about 25% of the level seen withheat or light. To reduce this, the HSP was linked from −80 to +30 to theminimal pCMV promoter. The pCMV promoter is preferred due to its lowerbackground expression. Additionally, it permits greater amplification ofthe expression of the therapeutic gene, independent of the constraintsof the weaker HSP promoter, which is used to initiate the reaction witha burst of heat or light.

[0075] To further overcome the problem of background expression, twocassettes in the plasmid pDATH are introduced. An antisense to TET-On isplaced under the control of the pCMV promoter. The constitutivelyproduced antisense binds to any TET-On sense mRNA from the backgroundtranscription and prevents its being translated. An additional block onbackground transcription is provided in cassette #4 in which a dominantnegative TET-On with the DNA binding site, but not the transcriptionactivation domain, is placed under the control of the pCMV. This resultsin background transcription driving the production of TET-On anddominant negative TET-On, which then compete for the ptet binding site.

EXAMPLE 5

[0076] Monitoring of p53 Expression Levels

[0077] To ensure that a suitable level of antisense TET-On RNA anddominant negative TET-On protein is produced, levels of p53 expressionare monitored to calibrate copy number and strength of the promoterneeded in order to reduce background. First, cell lines harboring pDATHare isolated in the absence of tetracycline. The level of p53 or acotransfected ptet-EGFP is then monitored to determine the copy numberof antisense TET-On and dominant negative TET-On that needs to beincorporated into pDATH to reduce background expression.

EXAMPLE 6

[0078] The Expression Vector pRIBs for Treatment of Local and MetastaticBreast and Ovarian Cancer

[0079] As mentioned supra, genes placed under the control of suchpromoters as the radiation inducible promoter of the Egr-1 gene areoften expressed only transiently and at low levels. This renders themunsuitable for use in cancer therapy. To overcome these problems, theexpression vector pRIBs-X (Radiation-Inducible, Breast-specificPromoter) was designed.

[0080] Gene expression levels were optimized using a feed-forwardreaction with the tetracycline-dependent transactivator, Tet-On, placedunder the control of a tetracycline promoter (tetp), followed by theGAL-4 promoter (pGAL). Transient transcription initiated at pGAL leadsto synthesis of a low level of Tet-On, which then binds to tetp in thepresence of tetracycline. Tet-On then amplifies its own expression andthat of the therapeutic gene linked to it via a feed-forward reaction.The expression of therapeutic genes is controlled by six gene cassettesin the pRIBs vector (FIG. 3). In cassette 1, the fusion gene GAL-DBD-mx(HLH-LZ domain of max fused to the DNA-binding domain of GAL-4) isregulated by EGRp. Background expression of GAL-DBD-mx is suppressed bya constitutively expressed antisense GAL-DBD-mx and a dominant negativeGAL-DBD in cassette 2. In cassette 3, the transcription activationdomain of the herpes simplex viral protein VP16 is fused to the HLH-LZdomain of c-Myc. The resulting fusion gene, VP16-TA-mc, placed under thecontrol of the c-erbB-2 promoter, is expressed in breast tumor cellsoverexpressing c-erbB-2. GAL-DBD-mx fusion protein binds to andactivates transcription from the pGAL promoter (cassette 4) byrecruiting the VP16-TA-mc proteins.

[0081] In unirradiated cells, the translation of the backgroundGAL-DBD-mx mRNA is reduced and the dominant negative GAL-DBD (withoutmx) competitively occupies the GALp in cassette 4, blocking Tet-Onexpression. Upon irradiation, GAL-DBD-mx is transiently induced 3-4 foldand temporarily overcomes the suppression by cassette 2. The GAL-DBD-mxrecruits the VP-16-TA-mc (a fusion gene of the VP16 transactivationdomain and the leucine zipper of myc under the control of the c-erbB-2promoter) to the GALp and activates a low level of Tet-On transcriptionstarting the feed-forward reaction.

[0082] In a treatment scheme using pRIBs-TNFα, for example, can bedelivered systemically in a liposome complex or as a recombinant virusto tumor and normal cells alike. Without radiation and tetracycline,TNFα is not expressed. Oxytetracycline is then administered systemicallyfollowed by X-ray irradiation of known metastatic tumor sites. As aresult, TNFα expression is induced in the tumor sites by the X-ray andamplified and maintained by oxytetracycline. Even though not all tumorcells may take up pRIBs-TNFα, tumor cells in the vicinity of those thatdo are exposed to the very high local concentration of TNFα secreted.The design of pRIBs-TNFα confers TNFα expression in the breast tumorcells only and not in the irradiated normal cells that were in the pathof the X-ray. As such, systemic toxicity, if any, is limited to the lowlevel of TNFα diffused from the tumor cells. In addition to, or insteadof, TNFα, another therapeutic gene, designated X, can be used with thepRIBS vector.

[0083] The structure of pRIBs-GFP-1 is shown in FIG. 3 and the mode ofaction summarized in FIG. 5. In unirradiated cells, backgroundGAL-DBD-mx expression and function are suppressed by cassette 2 in twoways. The antisense to GAL-DBD-mx suppresses the translation ofbackground GAL-DBD-mx mRNA whereas the GAL-DBD protein acts as adominant negative inhibitor by competing with GAL-DBD-mx for the pGALpromoter. In irradiated cells, GAL-DBD-mx expression is transientlyinduced three to 4 fold, overcoming the suppression by cassette 2. TheGAL-DBD-mx recruits the VP-16-TA-mc (a fusion gene of the VP16transactivation domain and the leucine zipper of Myc under the controlof the c-erbB-2 promoter) to the GALp and activates the transientexpression of the transactivator TET-ON. In the presence oftetracycline, Tet-ON is activated and it binds to and transactivates thetetp promoter (Gossen, M., et al., Science, 268:1766-1769 (1995)),amplifying its own level and GFP in a feed-forward reaction. Backgroundexpression of TET-ON and GFP is null in the absence of radiation ortetracycline.

EXAMPLE 7

[0084] Generation of Cell Lines and Xenografts Stably ExpressingpRIBs-GFP

[0085] Two pRIBs-GFP plasmids, pRIBs-GFP-1 and pRIBs-GFP-4, with one andfour copies of antisense and dominant negative gene cassettes,respectively, were constructed and stably transfected into thefibrosarcoma cell line HTB152 and the breast tumor cell lines SK-BR-3and MDAMB231 for in vitro analysis. 5×10⁶ cells are xenografted intoSCID mice. While all three human cell lines form poorly differentiatedtumors, only SK-BR-3 expresses a high level of c-erbB-2. Indeed,anti-erbB-2 intracellular single-chain antibody which down-regulatescell surface erbB-2, induces apoptosis only in SK-BR-3 but notMDA-MB-231 (Chumakov A. M., et al., Oncogene 8:3005-3011 (1993)).

[0086] The pRIBs-GFP-1 and -4 plasmids are thus used as models tooptimize the conditions for testing treatment of metastatic breast tumorxenografts in nude mice with cytotoxic genes. As cytotoxic genes linkedto EGRp are induced only in irradiated cells, toxicity to unirradiatedcells is eliminated. However, it is important to prevent expression ofcytotoxic genes in normal cells that are in the pathway of the X-ray.The three cell lines, which differ in c-erbB-2 expression, show thatcontrolling VP16-TA-mc expression with a tissue- or tumor-specificpromoter confines expression to irradiated breast tumor cells only andnot the irradiated normal cells of the vital organs where the metastatictumor cells reside.

[0087] The pRIBs-GFP plasmids are assembled as shown in FIG. 3. TheGAL-DBA-mx and the VP16-TA-mc are modified from the mammalian two hybridsystem (Fearon, E. R., et al., Proc. Natl. Acad. Sci. USA, 89:7958-7962(1992)). Two plasmids, pRIBs-GFP-1 and pRIBs-GFP-4, with 1 and 4 copiesof antisense and dominant negative GAL-DBD driven by the minimal CMVpromoter are tested.

[0088] All three cell lines are cotransfected with pRIBs-GFP and a SVneoplasmid. Cell lines stably expressing pRIBs-GFP-1 and pRIBs-GFP-4 areisolated by selection in G418. For in vivo analysis, 5×10⁶ cells of eachof the cell lines stably expressing the pRIBs-GFP plasmids are implantedinto the flank of SCID mice (four per group) and allowed to grow to 0.5cm in diameter. The expression of GFP in vitro and in the xenograftswithout radiation or oxytetracycline is analyzed by extracting theproteins into EBC buffer from the pulverized tumor and the amount ofprotein is quantitated by RIA.

[0089] The inducible level of GFP in vitro is measured by Westernanalysis and quantitated by RIA after irradiating the cells at 0-4 Gywith a Varian Clinac 2000 X-ray generator followed by administration of0-2 μg/ml of oxytetracycline. Data using HSPp showed that thefeed-forward reaction is very efficient and 0.01 μg/ml is sufficient toinduce a nine-fold increase of p53 expression in 10 hours. For in vivoanalysis, tumors are exposed to 0-4 Gy/X-ray. Six hours after radiation,0-15 μg/g of oxytetracycline is injected intraperitoneally. At 3 hourintervals (for 24 hours) after an injection, tumor mass is removed andthe amount of TET-ON and GFP measured relative to the total amount ofactin proteins. To achieve a higher or lower level of GFP, theexperiments are repeated with the level of TET-ON modified by adjustingthe dose of oxytetracycline. The rate of oxytetracycline removal byexcretion is monitored by analyzing plasma concentration at three hourintervals.

EXAMPLE 8

[0090] Targeting Metastatic Breast Tumors With WAPp or ST3p

[0091] The c-erbB-2 promoter had been chosen to initially validate thepRIB-X concept because human cancers overexpressing c-erbB-2 areassociated with poor prognosis. It is unlikely, however, that oneparticular promoter will address the problem of treating differentbreast tumors. Therefore it is also important to target GAL-DBD-mxexpression to metastatic breast tumors with the whey acidic proteinpromoter, WAPp (McKnight, R. A., et al., Mol. Reprod. & Dev., 44:179-184(1996)) or the stromelysin 3 promoter, ST3p (Ahmad, A., et al., Int. J.of Cancer, 73:290-296 (1997)). WAPp targets expression to breastepithelial cells while ST3p targets expression tomatrix-metalloproteinase-secreting stromal cells adjacent to tumors.

[0092] pRIBs is reconstructed by replacing the c-erbB-2 promoter witheither WAPp or ST3p. Breast and other tumor cell lines are screened forhigh and low expression of WAP and ST3. Cell lines differing in theirexpression of WAP and/or ST3 are used to test the expression of GFP.

[0093] The WAP promoter has been shown to be very specific for lactatingmammary epithelial cells in transgenic animals (Tzeng Y J., et al.,Oncogene 16(16):2103-2114 (1998)) and the stromelysin 3 promoter, ST3p,has been shown to be expressed only in stromal fibroblasts adjacent tocancer cells. Evidence suggests that production in stromal cell ofmatrix-metalloproteinases (including ST3), implicated in the process oftumor metastasis, is stimulated by the cancer cells. Thus, the targetingof VP16-TA-mc to the stromal cells will lead to the expression andrelease of therapeutic gene products in the vicinity of the metastatictumor cells. It must be noted that additional treatment specificity isattained by delivering pRIBs-X with liposomes coated with antibodies toc-erbB2.

EXAMPLE 9

[0094] The Expression Vector pRIPs for Treatment of Local and MetastaticProstate Cancer

[0095] As mentioned supra, genes placed under the control of suchpromoters as the radiation inducible promoter of the Egr-1 gene areoften expressed only transiently and at low levels. This renders themunsuitable for use in cancer therapy. To overcome these problems, theexpression vector pRIPs-X (Radiation-Inducible, Prostate-specificPromoter) was designed.

[0096] The pRIPS vector is comprised of six cassettes. Gene cassette 1differs from previously described vectors only in that it contains“Gal-DBD-mx” which is a fusion ORF encoding the N-terminus (amino acids1-147) DNA-binding domain of the yeast GAL4 protein (Gal-DBD) fused tothe basis helix-loop-helix-leucine zipper (bHLHLZ) domain of Max (mx,amino acids 8-112) followed by SV40 poly A. Gene cassette 2 is comprisedof the minimal CMV promoter (mCMVp), “antisense Gal-DBD-mx”, which is anantisense construct complementary to the Gal-DBD-mx sequence, “IRES”,which is an internal ribosomal entry site and “Gal-DBD” which competeswith the Gal-DBD-mx for the pGAL binding site. Gene cassette 3 iscomprised of “VP16-TA-mc” which is a fusion ORF encoding at theN-terminus the first 11 amino acids of Gal4 (amino acids 1-147),followed by the nuclear localization signal of the SV40 large T antigen,the 130 amino acid C-terminus transactivation domain of the herpessimplex viral protein VP16, the bHLHLZ domain of c-Myc (amino acids350-439), followed by SV40 polyA. The resulting fusion gene, VP16-TA-mc,is placed under the control of the probasin gene promoter “pProbasin” upto the first ATG. Gene cassette 4 contains “GALp”, consisting of fivecopies of a 17-mer DNA-binding site for Gal4. The TET-ON sequence isplaced under the control of the GALp-ptet promoter and the therapeuticgene, X, is linked to the TET-ON via an IRES; Gene cassette 5 containsan antisense TET-ON which is a sequence consisting of the complementarysequence to the first 80 bases of the TET-ON sequence including the ATG,placed under the control of the pCMV promoter. Gene cassette 6 containsa dominant negative TET-ON consisting of the coding sequences for aminoacids 1-207 of the tet repressor placed under the control of the pCMVpromoter. In other variants of pRIPs-X, pProbasin is replaced by PSA,the promoter region of the prostate specific antigen, or otherprostate-specific genes.

EXAMPLE 10

[0097] The Expression Vector pHIBs-X for Treatment of Local andMetastatic Breast and Ovarian Cancer

[0098] The expression vector pHIBs-X was designed and is comprised ofsix cassettes. Gene cassette 1 differs from previously described vectorsonly in that it contains “Gal-DBD-mx” which is a fusion ORF encoding theN-terminus (amino acids 1-147) DNA-binding domain of the yeast GAL4protein (Gal-DBD) fused to the basis helix-loop-helix-leucine zipper(bHLHLZ) domain of Max (mx, amino acids 8-112) followed by SV40 poly A.The resulting fusion gene GAL-DBD-mx is controlled by the heat inducibleHSP promoter. Gene cassette 2 is comprised of the minimal CMV promoter(mCMVp), “antisense Gal-DBD-mx”, which is an antisense constructcomplementary to the Gal-DBD-mx sequence, “IRES”, which is an internalribosomal entry site and “Gal-DBD” which competes with the Gal-DBD-mxfor the pGAL binding site. Gene cassette 3 is comprised of “VP16-TA-mc”which is a fusion ORF encoding at the N-terminus the first 11 aminoacids of Gal4 (amino acids 1-147), followed by the nuclear localizationsignal of the SV40 large T antigen, the 130 amino acid C-terminustransactivation domain of the herpes simplex viral protein VP16, thebHLHLZ domain of c-Myc (amino acids 350-439), followed by SV40 polyA.The resulting fusion gene, VP-16TA-mc, is placed under the control ofthe c-erbB-2 promoter “perbB2” up to the first ATG. Gene cassette 4contains “GALp”, consisting of five copies of a 17-mer DNA-binding sitefor Gal4. The TET-ON sequence is placed under the control of theGALp-ptet promoter and the therapeutic gene, X, is linked to the TET-ONvia an IRES; Gene cassette 5 contains an antisense TET-ON which is asequence consisting of the complementary sequence to the first 80 basesof the TET-ON sequence including the ATG, placed under the control ofthe pCMV promoter. Gene cassette 6 contains a dominant negative TET-ONconsisting of the coding sequences for amino acids 1-207 of the tetrepressor placed under the control of the pCMV promoter.

[0099] The pHIBs-X expression vector is identical to the pRIBs-X plasmidexcept for gene cassette 1 where the Egr-1 promoter in pRIBs-X isreplaced by the HSP 70 promoter. pHIBs-X specifically targets local andmetastatic breast and ovarian tumors when the tumors are exposed toheat.

EXAMPLE 11

[0100] The Expression Vector pHIPs-X for Treatment of Local andMetastatic Prostate Cancer

[0101]FIG. 10 illustrates the structure of the pHIPs-GFP(Heat-Inducible, Prostate-specific Promoter) expression vector. Thisvector is comprised of six cassettes. Gene cassette 1 differs frompreviously described vectors only in that it contains “Gal-DBD-mx” whichis a fusion ORF encoding the N-terminus (amino acids 1-147) DNA-bindingdomain of the yeast GAL4 protein (Gal-DBD) fused to the basishelix-loop-helix-leucine zipper (bHLHLZ) domain of Max (mx, amino acids8-112) followed by SV40 poly A. The resulting fusion gene GAL-DBD-mx iscontrolled by the heat inducible HSP promoter. Gene cassette 2 iscomprised of the minimal CMV promoter (mCMVp), “antisense Gal-DBD-mx”,which is an antisense construct complementary to the Gal-DBD-mxsequence, “IRES”, which is an internal ribosomal entry site and“Gal-DBD” which competes with the Gal-DBD-mx for the pGAL binding site.Gene cassette 3 is comprised of “VP16-TA-mc” which is a fusion ORFencoding at the N-terminus the first 11 amino acids of Gal4 (amino acids1-147), followed by the nuclear localization signal of the SV40 large Tantigen, the 130 amino acid C-terminus transactivation domain of theherpes simplex viral protein VP16, the bHLHLZ domain of c-Myc (aminoacids 350-439), followed by SV40 polyA. The resulting fusion gene,VP16-TA-mc, is placed under the control of the probasin gene promoter(pProbasin) up to the first ATG. Gene cassette 4 contains “GALp”,consisting of five copies of a 17-mer DNA-binding site for Gal4. TheTET-ON sequence is placed under the control of the GALp-ptet promoterand the therapeutic gene, X, is linked to the TET-ON via an IRES; Genecassette 5 contains an antisense TET-ON which is a sequence consistingof the complementary sequence to the first 80 bases of the TET-ONsequence including the ATG, placed under the control of the pCMVpromoter. Gene cassette 6 contains a dominant negative TET-ON consistingof the coding sequences for amino acids 1-207 of the tet repressorplaced under the control of the pCMV promoter.

[0102] The pHIPs-X expression vector is identical to the pRIPs-X plasmidexcept for gene cassette 1 where the Egr-1 promoter in pRIBs-X andpRIPs-X is replaced by the HSP 70 promoter. pHIPs-X specifically targetslocal and metastatic prostate tumors when the tumors are exposed toheat.

[0103] Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

[0104] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

What is claimed is:
 1. A method of achieving localized, temporalexpression of a gene under control of a heat inducible promoter,comprising the steps of: inserting said gene into a cloning site of apDATH-X (Dominant negative, Antisense, TET-ON controllable Heat shockpromoter plasmid) vector, said vector comprising: a) cassette 1comprising TET-ON expressed under the control of a heat shock promoterand a tet operator, wherein said TET-ON consists of a fusion of thecoding sequences for amino acids 1-207 of tetracycline repressor and theC-terminus last 130 amino acid transcription activation domain of VP16protein of the herpes simplex virus, wherein said heat shock promoterconsists of heat shock response elements (−260 to 30) of the human heatshock 70 gene promoter linked to a minimal cytomegalovirus promoter,pCMV; wherein said tet operator consists of 19 base pair invertedrepeats of operator O2 of TN10 to which said tet repressor and TET-ONbind; b) cassette 2 comprising a cloning site for a therapeutic genedownstream of a tetp-CMV promoter consisting of a tet operator linked toa minimal cytomegalovirus promoter, pCMV, wherein said tet operatorconsists of 19 base pair inverted repeats of operator O2 of TN10 towhich said tet repressor and TET-ON bind; c) cassette 3 comprisingantisense TET-ON under the control of pCMV promoter, wherein saidantisense TET-ON consists of an antisense sequence complementary to thefirst 80 nucleotides of the TET-ON sequence including the ATG startcodon; and d) cassette 4 comprising a dominant negative TET-ON under thecontrol of pCMV promoter, wherein said dominant negative TET-ON consistsof a tet repressor without a VP16 transactivation domain; introducingthe vector containing said gene into the host organism; and applyingheat energy to a location on said host organism where expression of saidgene is desired.
 2. The method of claim 1, where said host organism is ahuman.
 3. A recombinant vector, pDATE-X (Dominant negative, Antisense,TET-ON controllable EGR promoter expression plasmid), said vectorcomprising the cassettes: (a) cassette 1 comprising the TET-ON sequenceunder the control of the EGRp, the tetracycline operator binding siteand pCMV; (b) cassette 2 comprising a therapeutic gene X under thecontrol of the tetp-pCMV promoter; (c) cassette 3 comprising antisenseTET-ON under the control of the pCMV promoter; and (d) cassette 4comprising dominant negative TET-ON under the control of the pCMVpromoter.
 4. A recombinant vector, pRIBs-X, (Radiation-Inducible,Breast-specific Promoter) expression vector, said vector comprising thecassettes: (a) cassette 1 comprising “Gal-DBD-mx” which is a fusion openreading frame encoding the N-terminus (amino acids 1-147) DNA-bindingdomain of the yeast GAL4 protein (Gal-DBD) fused to the basichelix-loop-helix-leucine zipper domain of Max (amino acids 8-112)followed by SV40 poly A, wherein the resulting fusion gene GAL-DBD-mx iscontrolled by the radiation inducible Egr-1 promoter; (b) cassette 2comprising the minimal CMV promoter, “antisense Gal-DBD-mx”, which is anantisense construct complementary to the Gal-DBD-mx sequence, aninternal ribosomal entry site (IRES) and “Gal-DBD” which competes withthe Gal-DBD-mx for the pGAL binding site; (c) cassette 3 comprising“VP16-TA-mc” which is a fusion ORF encoding at the N-terminus the first11 amino acids of Gal4 (amino acids 1-147), followed by the nuclearlocalization signal of the SV40 large T antigen, the 130 amino acidC-terminus transactivation domain of the herpes simplex viral proteinVP16, the basic helix-loop-helix-leucine zipper domain of c-Myc (aminoacids 350-439), followed by SV40 polyA, wherein the resulting fusiongene, VP16-TA-mc, is under the control of the c-erbB2 promoter “perB2”up to the first ATG; (d) cassette 4 comprising “Galp”, five copies of a17-mer DNA-binding site for Gal4, wherein a TET-ON sequence is placedunder the control of the GAPp-ptet promoter and a therapeutic gene X islinked to the TET-IN via an IRES; (e) cassette comprising an antisenseTET-ON which is a sequence consisting of the complementary sequence tothe first 80 bases of the TET-ON sequence including the ATG under thecontrol of the pCMV promoter; and (f) cassette 6 comprising a dominantnegative TET-ON consisting of the coding sequences for amino acids1-207.
 5. The recombinant vector of claim 4, wherein the perbB2 promoterof cassette 3 is replaced with the whey acidic protein promoter.
 6. Therecombinant vector of claim 4, wherein the perbB2 promoter of cassette 3is replaced with the stromelysin 3 promoter.
 7. The recombinant vectorof claim 4, wherein said gene X is a gene encoding tumor necrosis factoralpha.
 8. A method of treating local and metastatic breast and ovariancancer comprising the step of: administering the expression vector ofclaim 4 to an individual in need of such treatment.
 9. A method oftreating local and metastatic breast and ovarian cancer comprising thestep of: administering the expression vector of claim 4 to an individualin need of such treatment.
 10. A method of treating local and metastaticbreast and ovarian cancer comprising the step of: administering theexpression vector of claim 6 to an individual in need of such treatment.11. A recombinant pRIPs-X (Radiation-Inducible, Prostate-specificPromoter) expression vector, said vector comprising the cassettes: (a)cassette 1 comprising “Gal-DBD-mx” which is a fusion open reading frameencoding the N-terminus (amino acids 1-147) DNA-binding domain of theyeast GAL4 protein fused to the basic helix-loop-helix leucine zipperdomain of Max (amino acids 8-112) followed by SV40 polyA, wherein theresulting fusion gene GAL-DBD-mx is controlled by the radiationinducible Egr-1 promoter; (b) cassette 2 comprising the minimal CMVpromoter, antisense Gal-DBD-mx, which is an antisense constructcomplementary to the Gal-DBD-mx sequence, IRES, which is an internalribosomal entry site and Gal-DBD which competes with the Gal-DBD-mx forthe pGAL binding site; (c) cassette 3 comprising “VP16-TA-mc”, a fusionopen reading frame encoding at the N-terminus the first 11 amino acidsof Gal4, followed by the nuclear localization signal of the SV40 large Tantigen, the 130 amino acid C-terminus transactivation domain of theherpes simplex viral protein VP16, the basic helix-loop-helix leucinezipper domain of c-Myc (amino acids 350-439), followed by SV40 polyA,wherein the resulting fusion gene, VP16-TA-mc, is under the control ofthe probasin gene promoter “pProbasin” up to the first ATG; (d) cassette4 comprising GALp, five copies of the 17-mer DNA-binding site for Gal4,wherein the TET-ON sequence is under the control of the GALp-ptetpromoter and a therapeutic gene X is linked to the TET-ON via aninternal ribosomal entry site; (e) cassette 5 comprising an antisenseTET-ON which is a sequence consisting of the complementary sequence tothe first 80 bases of the TET-ON sequence including the ATG, under thecontrol of the pCMV promoter; and (f) cassette 6 comprising a dominantnegative TET-ON consisting of the coding sequence for amino acids 1-207.12. The recombinant vector of claim 11, wherein said probasin promoterof cassette 3 is replaced with the prostate specific antigen promoter.13. The recombinant vector of claim 11, wherein said gene X is tumornecrosis factor alpha.
 14. A method of treating local and metastaticprostate cancer comprising the step of: administering the expressionvector of claim 11 to an individual in need of such treatment.
 15. Amethod of treating local and metastatic prostate cancer comprising thestep of: administering the expression vector of claim 12 to anindividual in need of such treatment.
 16. A recombinant expressionvector, pHIBs-X (Heat Inducible, Breast-specific promoter), said vectorcomprising the cassettes: (a) cassette 1 comprising Gal-DBD-mx which isa fusion open reading frame encoding the N-terminus (amino acids 1-147)DNA-binding domain of the yeast GAL4 protein fused to the basichelix-loop-helix leucine zipper domain of Max (amino acids 8-112)followed by SV40 polyA, wherein the resulting fusion gene GAL-DBD-mx iscontrolled by the heat inducible heat shock protein promoter; (b)cassette 2 comprising the minimal CMV promoter, antisense Gal-DBD-mx, aconstruct complementary to the Gal-DBD-mx sequence, an internalribosomal entry site and Gal-DBD, which competes with the Gal-DBD-mx forthe pGAL binding site; (c) cassette 3 comprising “VP16-TA-mc” which is afusion open reading frame encoding at the N-terminus the first 11 aminoacids (amino acids 1-147), followed by the nuclear localization signalof the SV40 large T antigen, the 130 amino acid C-terminustransactivation domain of the herpes simplex viral protein VP16, thebasic helix-loop-helix leucine zipper domain of c-Myc (amino acids350-439), followed by SV40 polyA, wherein the resulting fusion geneVP16-TA-mc is under the control of the c-erbB2 gene promoter “perbB2” upto the first ATG; (d) cassette 4 contains GALp, five copies of a 17-merDNA-binding site for Gal4, wherein the TET-ON sequence is under thecontrol of the GALp-ptet promoter and a therapeutic gene, X, is linkedto the TET-ON via an internal ribosomal entry site; (e) cassette 5comprising an antisense TET-ON which is a sequence consisting of thecomplementary sequence to the first 80 bases of the TET-ON sequenceincluding the ATG, under the control of the pCMV promoter; and (f)cassette 6 comprising a dominant negative TET-ON consisting of thecoding sequences for amino acids 1-207.
 17. The recombinant vector ofclaim 16, wherein the perbB2 promoter of cassette 3 is replaced with thewhey acidic protein promoter.
 18. The recombinant vector of claim 16,wherein the perbB2 promoter of cassette 3 is replaced with thestromelysin 3 promoter.
 19. The method of claim 16, wherein saidtherapeutic gene is tumor necrosis factor alpha.
 20. A method oftreating local and metastatic breast and ovarian cancer comprising thestep of: administering the expression vector of claim 16 to anindividual in need of such treatment.
 21. A method of treating local andmetastatic breast and ovarian cancer comprising the step of:administering the expression vector of claim 17 to an individual in needof such treatment.
 22. A method of treating local and metastatic breastand ovarian cancer comprising the step of: administering the expressionvector of claim 18 to an individual in need of such treatment.
 23. Arecombinant vector, pHIPs-X (Heat-Inducible, Prostate-specificPromoter), said vector comprising the cassettes: (a) cassette 1comprising Gal-DBD-mx which is a fusion open reading frame encoding theN-terminus (amino acids 1-147) DNA-binding domain of the yeast GAL4protein fused to the basic helix-loop-helix leucine zipper domain of Max(amino acids 8-112) followed by SV40 polyA, wherein the resulting fusiongene GAL-DBD-mx is controlled by the heat inducible heat shock proteinpromoter; (b) cassette 2 comprising the minimal CMV promoter (mCMVp),antisense Gal-DBD-mx, a construct complementary to the Gal-DBD-mxsequence, an internal ribosomal entry site and Gal-DBD, which competeswith the Gal-DBD-mx for the pGAL binding site; (c) cassette 3 comprising“VP16-TA-mc”, a fusion open reading frame encoding at the N-terminus thefirst 11 amino acids of Gal4, followed by the nuclear localizationsignal of the SV40 large T antigen, the 130 amino acid C-terminustransactivation domain of the herpes simplex viral protein VP16, thebasic helix-loop-helix leucine zipper domain of c-Myc (amino acids350-439), followed by SV40 polyA, wherein the resulting fusion gene,VP16-TA-mc, is under the control of the probasin gene promoter“pProbasin” up to the first ATG; (d) cassette 4 comprising GALp, fivecopies of a 17-mer DNA-binding site for Gal4, wherein the TET-ONsequence is under the control of the GALp-ptet promoter and atherapeutic gene, X, is linked to the TET-ON via an internal ribosomalentry site; (e) cassette 5 comprising an antisense TET-ON which is asequence consisting of the complementary sequence to the first 80 basesof the TET-ON sequence including the ATG, under the control of the pCMVpromoter; and (f) cassette 6 comprising a dominant negative TET-ONconsisting of the coding sequences for amino acids 1-207.
 24. Therecombinant vector in claim 23, wherein the probasin promoter isreplaced with the prostate-specific antigen promoter.
 25. Therecombinant vector of claim 23, wherein said therapeutic gene is tumornecrosis alpha.
 26. A method of treating local and metastatic prostatecancer comprising the step of: administering the expression vector ofclaim 23 to an individual in need of such treatment.
 27. A method oftreating local and metastatic prostate cancer comprising the step of:administering the expression vector of claim 25 to an to individual inneed of such treatment.